Purpose: :
Long-lived proteins, including those found in the lens, undergo numerous modifications as a result of prolonged exposure to physiological conditions, however the details of several of these reactions remain to be elucidated. These age-related modifications contribute to deterioration in health and fitness. One such major modification is racemisation, although the mechanism is not well understood.

Methods: :
Peptides were exposed to prolonged incubation at 37°C, as well as to elevated temperatures, at pH 7 and the products characterised using MALDI mass spectrometry, ESI mass spectrometry and NMR spectroscopy. One peptide examined, PFHSPS, is based on a sequence known to be modified in human αB crystallin. All peptides had a C-Terminal Tyr added to aid detection by HPLC.

Results: :
Incubation of peptides PFHSPSY, PAHSPSY, PEHSPSY, PKHSPSY, resulted in facile racemisation of the N-terminal amino acid. Typically between 15 and 40% racemisation was observed after 2 weeks at 60°C.Incubating PFHSPSY under physiological conditions (37°C) resulted in ~5% racemisation after 8 weeks. AFHSPSY showed less N-terminal racemisation. Other modifications, for example, truncation of the peptides by loss of amino acid residues from the N-terminus were also observed.

Conclusions: :
Some reactions of long-lived proteins can be mimicked using exposure of peptides to elevated temperatures. The ramifications of N-terminal racemisation for protein structure and function are as yet unknown, although its impact will probably vary depending on the particular protein. In terms of susceptibility to enzymatic proteolysis, it is likely that having a D-amino acid at the N-terminus will inhibit cleavage of long-lived proteins by exopeptidases.